Identifying viable Neisseria gonorrhoeae through validation and application of viability RT-PCR

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ABSTRACT Gonorrhea, caused by Neisseria gonorrhoeae (NG), is rising globally alongside increasing antimicrobial resistance, including to first-line treatment. Standard diagnostics, such as nucleic acid amplification tests, cannot distinguish viable from non-viable bacteria, while culture—an inherent viability assessment—is challenged by the organism’s fastidious nature and environmental sensitivity. Molecular viability assays have not been applied to NG. This study aims to validate and apply a viability PCR (V-PCR) method for NG and perform an exploratory application on clinical samples to assess concordance with culture. Viability was assessed using PMAxx, a membrane-impermeable dye that binds nucleic acids in non-viable cells, enabling discrimination from viable cells. Technical validation used mixtures of viable and heat-killed NG with paired PMAxx-treated/PMAxx-untreated aliquots and subsequent porA qPCR to derive ΔCt values. In an exploratory application, clinical samples (anorectal, oropharyngeal, and urogenital) were tested using V-PCR and compared with culture. V-PCR eliminated 99.998% of DNA from non-viable NG, with a strong correlation ( R ²=0.96) across dilution series. Culture-positive clinical samples had a higher median viable load (3.92 log 10 copies/mL, interquartile range [IQR]: 1.71–5.23) than culture-negative samples (0.87 log 10 copies/mL, IQR: 0.38–2.69, P = 0.0003). Median viability percentage was significantly higher in culture-positive samples (16.92%, IQR: 0.41%–42.45%) versus culture-negative samples (1.23%, IQR: 0.00%–9.38%, P = 0.01). Overall, V-PCR reliably discriminated viable from non-viable NG in controlled experiments. In an exploratory application on clinical specimens, higher viable loads and viability percentages were associated with culture positivity. These findings support further evaluation of V-PCR as a tool to study factors influencing culture recovery; formal validation would be required before clinical implementation. IMPORTANCE Gonorrhea affects an estimated 82 million people annually, with rising antimicrobial resistance threatening its treatability. Accurate diagnostics are essential, yet a diagnostic gap persists: standard PCR cannot distinguish viable from non-viable Neisseria gonorrhoeae , while culture—an inherent viability assessment—is challenged by the organism’s fastidious nature and environmental sensitivity. This limits accurate viability assessment, which is relevant to diagnostic practice, antimicrobial resistance surveillance, and broader research into gonococcal infection. PMAxx-based viability PCR (V-PCR) has shown promise in other pathogens, including Chlamydia trachomatis and SARS-CoV-2, but had not been applied to N. gonorrhoeae . Here, V-PCR is validated for N. gonorrhoeae for the first time, demonstrating strong discrimination between viable and non-viable organisms with consistent performance across predefined viability ratios, and in an exploratory clinical application, viable load and viability percentage were significantly higher in culture-positive specimens. These findings establish a foundation for more accurate viability assessment in gonococcal diagnostics and research.

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